Saccharomyces cerevisiae bred by space breeding technology and mutation site application thereof

A technology of Saccharomyces cerevisiae and recombinant Saccharomyces cerevisiae, applied in the field of microorganisms, can solve the problems of reducing carotenoid synthesis efficiency, growth retardation and the like

Pending Publication Date: 2022-01-04
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It exhibits high carotenoid synthesis ability, but it has severe growth

Method used

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  • Saccharomyces cerevisiae bred by space breeding technology and mutation site application thereof
  • Saccharomyces cerevisiae bred by space breeding technology and mutation site application thereof
  • Saccharomyces cerevisiae bred by space breeding technology and mutation site application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1 microgravity mutagenesis

[0024] The Saccharomyces cerevisiae fermentation medium of the present embodiment is: YPD medium: yeast extract 10g / L (OXOID company), peptone 20g / L, glucose 20g / L, solvent is water; Or YPM medium: yeast extract 10g / L L (Angel Yeast Co., Ltd.), peptone 20g / L, glucose 20g / L, potassium dihydrogen phosphate 10g / L, magnesium sulfate heptahydrate 5g / L, potassium sulfate 3.5g / L, sodium phosphate 2.5g / L, TMS Solution 1ml / L (magnesium chloride hexahydrate 250mg / L, calcium chloride dihydrate 104.5mg / L, mg / L, copper sulfate pentahydrate 0.4mg / L, sodium iodide 0.08mg / L, manganese chloride tetrahydrate 0.1mg / L, sodium molybdate dihydrate 0.5mg / L, boric acid 1mg / L, cobalt chloride hexahydrate 0.3mg / L, zinc sulfate heptahydrate 6.25mg / L, ferrous sulfate heptahydrate 3.5mg / L), the solvent is water. Its preparation method is to mix each component uniformly and sterilize to prepare. The solid medium is based on the liquid medium by adding 2% a...

Embodiment 2

[0027] Embodiment 2: Further modification of W2 bacterial strain

[0028]In order to further enhance the carotenoid synthesis ability of S. cerevisiae W2 strain, we replaced the ACC1 gene promoter (its nucleotide sequence as shown in SEQ ID NO.1) with PDC1 (its nucleotide sequence) on the basis of Saccharomyces cerevisiae W2 strain The sequence is shown in SEQ ID NO.2), references (Xie ZX, et al. Rapid and Efficient CRISPR / Cas9-Based Mating-Type Switching of Saccharomyces cerevisiae.8,173-183(2018).) and construct diploid cells Strain W2-A-5 was obtained. It was deposited in Guangdong Microbial Culture Collection Center (GDMCC) on December 3, 2020, address: 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, postcode 510070, and the deposit number is: GDMCC No :61337.

[0029] Streak the obtained strains in fresh YPD solid medium respectively. After 48 hours, pick a single colony and inoculate it in 5ml of YPD l...

Embodiment 3

[0034] Example 3 Determination of the Mutation Site of the W2 Strain

[0035] In order to determine the mutation site of the mutant strain W2, we performed genome resequencing on the starting strain-S. Four single-nucleotide mutations were found, one of which was meaningful and produced a stop codon inside the CHO2 gene, resulting in gene inactivation. Subsequently, we carried out reverse metabolic engineering research on the starting strain, and used the CRISPR / Cas9 system to knock out the CHO2 gene (Gene ID: 853061) of the BL03-E-10 strain to obtain the CHO2 gene knockout mutant strain BL03-2A-1 .

[0036] Primers used in the construction of BL03-2A-1 and BL03-D-5 strains

[0037]

[0038] Inoculate BL03-E-10, Saccharomyces cerevisiae W2, and the modified strain BL03-2A-1 into YPM medium according to the method of Example 1, and culture them at 30°C and 200rpm for 96 hours, measure the carotenoid content in cells, and calculate As a percentage of dry weight, the result...

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Abstract

The invention discloses saccharomyces cerevisiae bred by a space breeding technology and mutation site application thereof. The saccharomyces cerevisiae W2 is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No: 61335. The strain W2-A-5 is obtained through further modification on the basis of W2, and the preservation number of the strain W2-A-5 is GDMCC No:61337. The saccharomyces cerevisiae mutant strain W2 is obtained through microgravity mutagenesis screening, and the carotenoid content of the strain can reach 45 mg/g of dry cell weight under the shake flask fermentation condition. W2-A-5 is obtained through further transformation on the basis of the strain, and the content of carotenoid can reach 68 mg/g of dry cell weight. Through genome sequencing and reverse metabolic engineering research, CHO2 gene inactivation is determined to be a main factor for improving the synthesis capability of the W2 carotenoid. After CHO2 in the strain without growth retardation is inactivated, the carotenoid synthesis capability is remarkably improved, and a new modification target is provided for improving the carotenoid synthesis capability by directionally modifying the saccharomyces cerevisiae.

Description

technical field [0001] The invention relates to the technical field of microbes, in particular to a strain of Saccharomyces cerevisiae selected by space breeding technology and the application of mutation sites thereof. Background technique [0002] Carotenoids are a large class of colored natural pigments. Due to their strong antioxidant properties, they can be used in cosmetics, nutritional products, and antioxidants. Currently, chemical synthesis and natural extraction are the main ways to prepare carotenoids. The large-scale production of carotenoids is limited by factors such as the complexity of the structure, unstable supply of raw materials, residual toxic substances, and low yield. Saccharomyces cerevisiae itself cannot synthesize carotenoids, but genetic engineering of Saccharomyces cerevisiae for carotenoid synthesis has made great progress, and many reports have reached a very high production level. [0003] Laboratory Adaptive Evolution (ALE) is a technique th...

Claims

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Application Information

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IPC IPC(8): C12N1/18C12N15/01C12N15/81C12N9/88C12N15/31C12P23/00C12R1/865
CPCC12N1/18C12N15/01C12N15/81C12N9/88C07K14/395C12P23/00C12Y401/01001Y02E50/10
Inventor 朱红惠苏卜利李安章邓名荣冯广达
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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